Specificity toward a single reaction is a well-known characteristic of catalytic antibodies. However, contrary to convention, catalytic antibody 4B2 possesses the ability to efficiently catalyze two unrelated reactions: a Kemp elimination and an allylic isomerization of a β,γ-unsaturated ketone. To elucidate how this multifaceted antibody operates, mixed quantum and molecular mechanics calculations coupled to Monte Carlo simulations were carried out. The antibody was determined to derive its adaptability for the mechanistically different reactions through the rearrangement of water molecules in the active site into advantageous geometric orientations for enhanced electrostatic stabilization. In the case of the Kemp elimination, a general base, Glu L34, carried out the proton abstraction from the isoxazole ring of 5-nitro-benzisoxazole while water molecules delivered specific stabilization at the transition state. The role of water was found to be more pronounced in the allylic isomerization because the solvent actively participated in the stepwise mechanism. A rate-limiting abstraction of the a-proton from the β,γ-unsaturated ketone via Glu L34 led to the formation of a neutral dienol intermediate, which was rapidly reprotonated at the y-position via a solvent hydronium ion. Preferential channeling of H3O+ in the active site ensured a stereoselective proton exchange from the a- to the y-position, in good agreement with deuterium exchange NMR and HPLC experiments. Ideas for improved water-mediated catalytic antibody designs are presented. In a technical advancement, improvements to a recent polynomial fitting and integration technique utilizing free energy perturbation theory delivered greater accuracy and speed gains.
ASJC Scopus subject areas
- Physical and Theoretical Chemistry
- Surfaces, Coatings and Films
- Materials Chemistry